Plants of the hemp family Cannabis produce significant amounts of cannabinoids. The compound .DELTA..sup.9 -tetrahydrocannabinol (.DELTA..sup.9 -THC) is an important cannabinoid which produces psychotrophic effects attributed to marijuana, hashish, and hash oil. Human metabolism of .DELTA..sup.9 - THC results in microsomal oxidation through 11-hydroxy-.DELTA..sup.9 -THC to a series of polar metabolites with 11-nor-.DELTA..sup.9 -THC-9-carboxylic acid being a primary metabolite. The 11-nor-.DELTA..sup.9 -THC-9-carboxylic acid is further catabolized to an ester-linked glucuronide, 11-nor-.DELTA..sup.9 -THC-9-carboxylic acid glucuronide, that is excreted in urine and feces (see citation 1, infra).
Synthetic routes to .DELTA..sup.9 -THC and its metabolites have been described previously (1-10). Although several approaches to the synthesis of 11-nor-.DELTA..sup.9 -THC-9-methanol and 11-nor-.DELTA..sup.9 -THC-9-carboxylic acid (1-11) have been described, the published syntheses are either time-consuming, produce racemic products, or give low yields. Recently the inventors disclosed an improved convenient synthesis of 11-nor-.DELTA..sup.9 -THC-9-methanol (13). An enzymatic approach for synthesis of small amounts of impure 5'-hydroxy-.DELTA..sup.9 -THC and 11-nor-.DELTA..sup.9 -THC-9-methanol has also recently been disclosed (12).
THC glucuronide metabolites are present in biological fluids in vanishingly small amounts. These compounds are sensitive to electrophiles and extremely labile toward nucleophiles, making their preparation and isolation very difficult. The glucuronide carboxylic acid ester linkage is particularly labile to nucleophilic attack making chemical synthesis of this linkage extraordinarily difficult (14-16). Because of the lability of the THC-carboxylic acid linked glucuronides (THC glucuronides), it has not proven feasible to prepare sufficient quantities for use as assay calibrators or standards, or for use in the production of antibodies for immunoassays. THC glucuronides are a most useful class of therapeutic and diagnostic reagents and it would be highly advantageous to have a source of quantities of highly-purified THC glucuronides.
Administration or use of marijuana or other products of the Cannabis plant can be detected through the analysis of biological fluids, such as blood or urine, e.g., in assays for .DELTA..sup.9 -THC, .DELTA..sup.9 -THC-9-carboxylic acid, or .DELTA..sup.9 -THC-carboxylic acid glucuronide. Immunoassays are most widely used for this purpose (17-34), however, because of a significant number of false positive results, the immunoassay findings are frequently confirmed by gas chromatography-mass spectroscopy (GC-MS). GC-MS is useful for both confirmation and quantitation of THC (35-37). Deuterated derivatives of 11-nor-.DELTA..sup.9 -THC-9-carboxylic acid (THC carboxylic acid) are commonly used as internal standards for GC-MS. Several problems are associated with the use of these deuterated THC carboxylic acids as standards. First, the deuterated THC carboxylic acid derivatives are relatively difficult to prepare and are expensive. Second, the base peak of the commonly used methyl-derivative is at m/z 316 and although the peak for the natural THC metabolite is at m/z 313, there is also a confusing peak at 316. Third, current methods for detection of the THC glucuronide metabolite require hydrolysis of the THC glucuronide metabolite, (i.e., within the GC-MS matrix), and the fragmentation pattern of the hydrolyzed product is then compared with that of a THC carboxylic acid standard. Hydrolysis efficiency in biological samples is highly variable, and dependent upon complex matrix effects and container wall effects, so that recovery, and detection limits of the GC-MS assay vary from sample to sample. Fourth, use of THC carboxylic acid as a standard does not provide an internal standard (i.e., for quantifying hydrolyis and ionization efficiency) for assessing the accuracy, precision, and detection limits of the GC-MS assay. As a result of these problems, a THC sample that has tested positive in an immunoassay, may test negative in a GC-MS assay. In view of the foregoing and other problems associated with the use of 11-nor-.DELTA..sup.9 -THC-9-carboxylic acid it would be highly desirable to be able to eliminate false test results and solve problems relating to the lack of suitable THC analytical standards. Novel THC glucuronide analytical standards and calibrators for testing THC metabolites in biological fluids would be highly desirable.
Objects of the invention provide processes for chemical synthesis of THC glucuronides, (including 11-nor-.DELTA..sup.9 -THC-9-carboxylic acid glucuronide), that are useful as immunogens for evoking antibodies, and as assay standards and calibrators in assays for detection and quantitation of THC metabolites in biological fluids. Other objects of the invention provide deuterated THC glucuronides, (e.g. deuterated-11-nor-.DELTA..sup.9 -THC-9-carboxylic acid glucuronides), that are useful as internal standards and assay calibrators in GC-MS assays. Still other objects of the invention provide improved GC-MS assays using deuterated THC glucuronides with undeuterated THC glucuronides as internal standards to improve specificity, accuracy, precision, and detection limits of the GC-MS assays. Further objects of the invention provide methods for quantifying recovery of THC metabolites in biological samples that are useful on a sample-by-sample basis. The quantification of recovery allows the detection limits of the assay to be adjusted lower or higher until the desired precision is reached. Such methods allow detection of THC metabolites in biological fluids with greater precision, specificity, and sensitivity and with fewer false-negative test results.